Tetrahydrofolate, which serves as the coenzyme for several key reactions involved in cell replication, can be generated from: (a) folate or dihydrofolate via dihydrofolate reductase; (b) 5-methyltetrahydrofolate via methionine synthetase; and (c) 5-formyltetrahydrofolate (folinate) via an indirect pathway, the first stept of which is catalyzed by 5,10-methenyltetrahydrofolate synthetase. Methotrexate (MTX), a folate antagonist used extensively in cancer chemotherapy, blocks cell replication by inhibiting dihydrofolate reductase. Folate, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate and MTX utilize the same transport system for entry in to cells. The overall objectives of this program are to obtain information about the folate transport system, dihydrofolate reductase, methionine synthetase and methenyltetrahydrofolate synthetase. L1210 mouse leukemia cells provide the primary experimental system. Specific areas of investigation will include: Isolation and characterization of the membrane-associated binding protein responsible for the transport of folate compounds (5-methyltetrahydrofolate, 5-formyltetrahydrofolate, folate and MTX) in L1210 mouse leukemia cells, and role of the protein in substrate internalization; Structure and catalytic mechanism of dihydrofolate reductase from L1210 cells, with particular reference to multiple forms of the enzyme varying in affinity for MTX: Isolation and characterization of methionine synthetase from L1210 cells, role of the enzyme and its cobalamin cofactor in cell replication, and mechanism of the enzyme and its counterpart from E. coli K-12; Structure and mechanism of 5,10-methenyltetrahydrofolate synthetase (5-formyltetrahydrofolate cyclodehydrase) from L. casei, parallel studies with its counterpart from L1210 cells, and role of the latter enzyme in the rescue of MTX-inhibited cells by 5-formyltetrahydrofolate (folinate).